microRNA (miRNA) are a recently uncovered class of regulatory RNAs which help to regulate gene expression, and control cell function. Over 2,000 human miRNAs have been discovered. Many miRNAs have tumor suppressor or oncogenic functions, and miRNA dysregulation has been shown to play a key role oncogenesis, metastasis, and even chemoresistance. Although much has been learned about miRNA biology, as the number of new miRNA genes have been discovered it has become increasingly challenging to annotate relevant miRNA regulatory networks. A critical limitation has been the lack of high-throughput, biological approaches for genome-wide analysis of miRNA behavior. In this R33 proposal, we will develop and validate a new technology and methodology that can be used to: (1) Measure the activity of each and every miRNA within a cell, at single cell resolution, and (2) Identify optimal synthetic miRNA binding sites that can be used to improve the targeting of suicide vectors and oncolytic viruses being developed for cancer therapies. Our technology will have major utility for expanding our understanding of cancer biology, for generating tools to study and even track specific cancer cell subsets, such as cancer stem cells, for screening for miRNA modulating drugs, and for developing novel therapies that better target tumor cells for destruction.